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Mismatch Repair in Gamma-Proteobacteria

伽玛变形菌中的错配修复

基本信息

批准号：
10116421
负责人：
Richard Fishel
金额：
$ 32.76万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-15 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10116421
关键词：
Adenine Bacteria Binding Proteins Biochemical Biology Biophysics Cells Closure by clamp Complement Factor B Complex DNA DNA Single Strand Break DNA strand break Defect Diffusion Distant ESKAPE pathogens Enterobacter Escherichia coli Event Excision Excision Repair Gammaproteobacteria Gene Mutation Genes Genetic Recombination Genome Goals Hereditary Nonpolyposis Colorectal Neoplasms Human Image Imaging Device Imaging Techniques Imaging technology In Vitro Individual Kinetics Klebsiella Lead Link Location Maintenance Malignant Neoplasms Mechanics Methodology Methylation Mismatch Repair Modeling Molecular Motion Multi-Drug Resistance Mutation Nucleotides Organism Parents Pathway interactions Pattern Polymerase Process Property Proteins Reaction Resolution Role Single-Stranded DNA Site Slide Testing Therapeutic Time Visualization biophysical properties cancer predisposition clinically relevant environmental adaptation exodeoxyribonuclease gene repair genetic analysis helicase homologous recombination in vivo in vivo imaging innovation molecular imaging pathogenic bacteria real-time images recruit single molecule stoichiometry visual tracking

项目摘要

MISMATCH REPAIR IN γ-PROTEOBACTERIA PROJECT SUMMARY / ABSTRACT Mismatch repair (MMR) is a highly conserved genome maintenance process that primarily resolves polymerase misincorporation errors. Mutation of the MMR genes dramatically increase spontaneous mutation rates and have been linked to adaptation as well as associated multi-drug resistance in several pathogenic bacteria. MMR mutations in humans are the cause of the common human cancer predisposition Lynch syndrome as well as numerous sporadic cancers. MMR is an excision-resynthesis process that is initiated at a DNA strand break, which may be hundreds to thousands of nucleotides away from the mismatch. The fidelity of MMR depends on establishing the ssDNA break on the error-containing DNA strand. A subset of γ-proteobacteria, including E.coli and the ESKAPE pathogens Klebsiella and Enterobacter, recently evolved DNA adenine methylation (Dam) and MutH to introduce a ssDNA break onto the newly replicated strand containing a misincorporation error. An important distinction between bacteria that utilize the Dam/MutH MMR Pathway and all other organisms is that dam mutations are lethal in combination with mutation of several homologous recombination genes (synthetic lethality). Similarly, mutations of MMR excision components are lethal in combination with replication editing gene mutations. The mechanisms that lead to MMR-dependent synthetic lethality are largely hypothetical. Deterministic models have historically underpinned MMR mechanisms, where definite complexes and progressions are proposed to complete the biochemical events. Using newly developed single molecule- imaging techniques we showed that the initial steps of MMR rely on random DNA diffusion mechanics that are modulated by the two most highly conserved MMR proteins in terrestrial biology, MutS and MutL. This new application will test the hypothesis that the entire multi-component MMR excision process is Stochastic (fully governed by random processes; the complete opposite of Deterministic). Understanding such biochemical randomness should impact future research and therapeutic strategies targeting MMR. We propose to use real-time single molecule imaging in vitro and in vivo to resolve the mechanics of E.coli MMR and its role in synthetic lethality. The Specific Aims are: 1.) quantitative biophysical imaging of individual MMR component activities, 2.) visualization of the complete MMR excision reaction on single mismatched DNA molecules, and 3.) analysis of MMR component interactions in live cells.

γ-变形菌中的错配修复